Transmission of blaNDM in Enterobacteriaceae among animals, food and human.

Despite carbapenems not being used in animals, carbapenem-resistant Enterobacterales (CRE), particularly New Delhi metallo-ß-lactamase-producing CRE (NDM-CRE), are prevalent in livestock. Concurrently, the incidence of human infections caused by NDM-CRE is rising, particularly in children. Although a positive association between livestock production and human NDM-CRE infections at the national level was identified, the evidence of direct transmission of NDM originating from livestock to humans remains largely unknown. Here, we conducted a cross-sectional study in Chengdu, Sichuan Province, to examine the prevalence of NDM-CRE in chickens and pigs along the breeding-slaughtering-retail chains, in pork in cafeterias of schools, and in colonizations and infections from children's hospital and examined the correlation of NDM-CRE among animals, foods and humans. Overall, the blaNDM increases gradually along the chicken and pig breeding (4.70%/2.0%) -slaughtering (7.60%/22.40%) -retail (65.56%/34.26%) chains. The slaughterhouse has become a hotspot for cross-contamination and amplifier of blaNDM. Notably, 63.11% of pork from the school cafeteria was positive for blaNDM. The prevalence of blaNDM in intestinal and infection samples from children's hospitals was 21.68% and 19.80%, respectively. whole genome sequencing (WGS) analysis revealed the sporadic, not large-scale, clonal spread of NDM-CRE along the chicken and pig breeding-slaughtering-retail chain, with further spreading via IncX3-blaNDM plasmid within each stage of whole chains. Clonal transmission of NDM-CRE is predominant in children's hospitals. The IncX3-blaNDM plasmid was highly prevalent among animals and humans and accounted for 57.7% of Escherichia coli and 91.3% of Klebsiella pneumoniae. Attention should be directed towards the IncX3 plasmid to control the transmission of blaNDM between animals and humans.

A systematic review and meta-analysis of carbapenem resistance and its possible treatment options with focus on clinical Enterobacteriaceae: Thirty years of development in Pakistan.

Background: Carbapenem resistance is epidemic worldwide, these last resort antimicrobials are listed in the WHO 'watch group' with higher resistance potential. During the years 2017-18 Pakistan Antimicrobial Resistance Surveillance System reported an increase in carbapenem resistance. However, a comprehensive information on prevalence and molecular epidemiology of carbapenem resistance in Pakistan is not available. This systematic review and meta-analysis is aimed to report the current carbapenem resistance situation in Pakistan and its treatment options. Methods: In this systematic review and meta-analysis, we investigated the pooled prevalence (PPr) of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae by organizing available data, from Web of Science and PubMed by April 2, 2020, in various groups and subgroups including species, years, provinces, extended spectrum ß-lactamase production, clinical presentation, carbapenemase and metallo-ß-lactamase production, and New Delhi metallo-ß-lactamase (NDM) prevalence. Literature review was updated for the studies publisehd by December 07, 2023. Moreover, we descriptively reviewed the molecular epidemiology of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae in Pakistan. Lastly, we statistically explored different treatment options available for carbapenem resistant infections. We used R package 'metafor' for performing meta-analysis and influence diagnostics and determining treatment options. Results: From two academic databases Web of Science and PubMed we identified 343 studies. Eighty-eight studies were selected for the systematic review and meta-analysis. Seventy-four studies were selected for phenotypic analysis, 36 for genotypic analysis, and 31 for available treatment options. PPr-ID of 12% [0.12 (0.07, 0.16)] was observed for phenotypic carbapenem resistance in Enterobacteriaceae with more prevalence recorded in Klebsiella pneumoniae 24% [0.24 (0.05, 0.44)] followed by 9% [0.09 (-0.03, 0.20)] in Escherichia coli. During the last two decades we observed a striking increase in carbapenem resistance PPr i.e., from 0% [0.00 (-0.02, 0.03)] to 36% [0.36 (0.17, 0.56)]. blaNDM with PPr 15% [0.15 (0.06, 0.23)] in naive isolates was found to be the fundamental genetic determinant for carbapenem resistance in Enterobacteriaceae in Pakistan. Polymyxin B, colistin, tigecycline, and fosfomycin were identified as the suggested treatment options available for multidrug resistant infections not responding to carbapenems. Various studies reported carbapenem resistance from human, animal, and environment sources. Conclusion: In conclusion, we found that NDM-1 producing carbapenem resistant Enterobacteriaceae are increasing in Pakistan. Meta-analysis showed that metallo-ß-lactamases producing E. coli ST405 and K. pneumoniae sequence type11 are the major resistant clones. Number of reported studies in various subgroups and inconsistency in following CLSI guidelines are the potential limitations of this meta-analysis. A National antimicrobial resistance (AMR) surveillance strategy based on One Health is urgently needed to check any future AMR crisis in Pakistan.

Evolution of triclosan resistance modulates bacterial permissiveness to multidrug resistance plasmids and phages.

The horizontal transfer of plasmids has been recognized as one of the key drivers for the worldwide spread of antimicrobial resistance (AMR) across bacterial pathogens. However, knowledge remain limited about the contribution made by environmental stress on the evolution of bacterial AMR by modulating horizontal acquisition of AMR plasmids and other mobile genetic elements. Here we combined experimental evolution, whole genome sequencing, reverse genetic engineering, and transcriptomics to examine if the evolution of chromosomal AMR to triclosan (TCS) disinfectant has correlated effects on modulating bacterial pathogen (Klebsiella pneumoniae) permissiveness to AMR plasmids and phage susceptibility. Herein, we show that TCS exposure increases the evolvability of K. pneumoniae to evolve TCS-resistant mutants (TRMs) by acquiring mutations and altered expression of several genes previously associated with TCS and antibiotic resistance. Notably, nsrR deletion increases conjugation permissiveness of K. pneumoniae to four AMR plasmids, and enhances susceptibility to various Klebsiella-specific phages through the downregulation of several bacterial defense systems and changes in membrane potential with altered reactive oxygen species response. Our findings suggest that unrestricted use of TCS disinfectant imposes a dual impact on bacterial antibiotic resistance by augmenting both chromosomally and horizontally acquired AMR mechanisms.

Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum ß-lactamase- and carbapenemase-producing bacteria.

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum ß-lactamases (blaCTX-M-15) and carbapenemases (blaNDM, blaOXA-48-like and blaKPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines.

Interphylum dissemination of NDM-5-positive plasmids in hospital wastewater from Fuzhou, China: a single-centre, culture-independent, plasmid transmission study.

BACKGROUND: The global spread of plasmid-borne carbapenem resistance is an ongoing public health challenge; however, the nature of such horizontal gene transfer events among complex bacterial communities remains poorly understood. We examined the in-situ transfer of the globally dominant New Delhi metallo-ß-lactamase (NDM)-5-positive IncX3 plasmid (denoted pX3_NDM-5) in hospital wastewater to simulate a real-world, One Health antimicrobial resistance context. METHODS: For this transmission study, we tagged pX3_NDM-5 with the green fluorescent protein gene, gfp, using a CRISPR-based method and transferred the plasmid to a donor Escherichia coli strain. Bacteria were extracted from a hospital wastewater treatment plant (Fujian Provincial Maternity and Children's Hospital, Fuzhou, China) as the bacterial recipient community. We mixed this recipient community with the E coli donor strain carrying the gfp-tagged plasmid, both with and without sodium hypochlorite (NaClO) as an environmental stressor, and conducted several culture-based and culture-independent conjugation assays. The conjugation events were observed microscopically and quantified by fluorescence-activated cell sorting. We analysed the taxonomic composition of the sorted transconjugal pool by 16S rRNA gene amplicon sequencing and assessed the stability of the plasmid in the isolated transconjugants and its ability to transfer back to E coli. FINDINGS: We show that the plasmid pX3_NDM-5 has a broad host range and can transfer across various bacterial phyla, including between Gram-negative and Gram-positive bacteria. Although environmental stress with NaClO did not affect the overall plasmid transfer frequency, it reduced the breadth of the transconjugant pool. The taxonomic composition of the transconjugal pool was distinct from that of the recipient communities, and environmental stress modulated the permissiveness of some operational taxonomic units towards the acquisition of pX3_NDM-5. Notably, pX3_NDM-5 transconjugants included the Gram-positive pathogen Enterococcus faecalis, and the plasmid could subsequently be reconjugated back to E coli. These findings suggest that E faecalis could act as a natural shuttle vector for the wide dissemination of pX3_NDM-5 plasmids. INTERPRETATION: Our culture-independent conjugation model simulates natural environmental conditions and challenges the established theory that Gram-negative and Gram-positive bacteria rarely exchange clinically important plasmids. The data show that plasmids disseminate more widely across genera and phyla than previously thought. These findings have substantial implications when considering the spread of antimicrobial resistance across One Health sectors. FUNDING: The Laboratory of Lingnan Modern Agriculture Project, the National Natural Science Foundation of China, the Natural Science Foundation of Fujian Province of China, and the Outstanding Young Research Talents Program of Fujian Agriculture and Forestry University.

Plasmid-mediated colistin-resistance genes: mcr.

Colistin is regarded as a last-line drug against serious infections caused by multidrug-resistant Gram-negative bacterial pathogens. Therefore, the emergence of mobile colistin resistance (mcr) genes has attracted global concern and led to policy changes for the use of colistin in food animals across many countries. Currently, the distribution, function, mechanism of action, transmission vehicles, origin of mcr, and new treatment strategies against MCR-producing pathogens have been extensively studied. Here we review the prevalence, structure and function of mcr, the fitness cost and persistence of mcr-carrying plasmids, the impact of MCR on host immune response, as well as the control strategies to combat mcr-mediated colistin resistance.

Carriage and within-host diversity of mcr-1.1-harbouring Escherichia coli from pregnant mothers: inter- and intra-mother transmission dynamics of mcr-1.1.

Exchange of antimicrobial resistance genes via mobile genetic elements occur in the gut which can be transferred from mother to neonate during birth. This study is the first to analyse transmissible colistin resistance gene, mcr, in pregnant mothers and neonates. Samples were collected from pregnant mothers (rectal) and septicaemic neonates (rectal and blood) and analysed for the presence of mcr, its transmissibility, genome diversity, and exchange of mcr between isolates within an individual and across different individuals (not necessarily mother-baby pairs). mcr-1.1 was detected in rectal samples of pregnant mothers (n = 10, 0.9%), but not in neonates. All mcr-positive mothers gave birth to healthy neonates from whom rectal specimen were not collected. Hence, the transmission of mcr between these mother-neonate pairs could not be studied. mcr-1.1 was noted only in Escherichia coli (phylogroup A & B1), and carried few resistance and virulence genes. Isolates belonged to diverse sequence types (n = 11) with two novel STs (ST12452, ST12455). mcr-1.1 was borne on conjugative IncHI2 bracketed between ISApl1 on Tn6630, and the plasmids exhibited similarities in sequences across the study isolates. Phylogenetic comparison showed that study isolates were related to mcr-positive isolates of animal origin from Southeast Asian countries. Spread of mcr-1.1 within this study occurred either via similar mcr-positive clones or similar mcr-bearing plasmids in mothers. Though this study could not build evidence for mother-baby transmission but the presence of such genes in the maternal specimen may enhance the chances of transmission to neonates.

Global, regional, and national estimates of the impact of a maternal Klebsiella pneumoniae vaccine: A Bayesian modeling analysis.

BACKGROUND: Despite significant global progress in reducing neonatal mortality, bacterial sepsis remains a major cause of neonatal deaths. Klebsiella pneumoniae (K. pneumoniae) is the leading pathogen globally underlying cases of neonatal sepsis and is frequently resistant to antibiotic treatment regimens recommended by the World Health Organization (WHO), including first-line therapy with ampicillin and gentamicin, second-line therapy with amikacin and ceftazidime, and meropenem. Maternal vaccination to prevent neonatal infection could reduce the burden of K. pneumoniae neonatal sepsis in low- and middle-income countries (LMICs), but the potential impact of vaccination remains poorly quantified. We estimated the potential impact of such vaccination on cases and deaths of K. pneumoniae neonatal sepsis and project the global effects of routine immunization of pregnant women with the K. pneumoniae vaccine as antimicrobial resistance (AMR) increases. METHODS AND FINDINGS: We developed a Bayesian mixture-modeling framework to estimate the effects of a hypothetical K. pneumoniae maternal vaccine with 70% efficacy administered with coverage equivalent to that of the maternal tetanus vaccine on neonatal sepsis infections and mortality. To parameterize our model, we used data from 3 global studies of neonatal sepsis and/or mortality-with 2,330 neonates who died with sepsis surveilled from 2016 to 2020 undertaken in 18 mainly LMICs across all WHO regions (Ethiopia, Kenya, Mali, Mozambique, Nigeria, Rwanda, Sierra Leone, South Africa, Uganda, Brazil, Italy, Greece, Pakistan, Bangladesh, India, Thailand, China, and Vietnam). Within these studies, 26.95% of fatal neonatal sepsis cases were culture-positive for K. pneumoniae. We analyzed 9,070 K. pneumoniae genomes from human isolates gathered globally from 2001 to 2020 to quantify the temporal rate of acquisition of AMR genes in K. pneumoniae isolates to predict the future number of drug-resistant cases and deaths that could be averted by vaccination. Resistance rates to carbapenems are increasing most rapidly and 22.43% [95th percentile Bayesian credible interval (CrI): 5.24 to 41.42] of neonatal sepsis deaths are caused by meropenem-resistant K. pneumoniae. Globally, we estimate that maternal vaccination could avert 80,258 [CrI: 18,084 to 189,040] neonatal deaths and 399,015 [CrI: 334,523 to 485,442] neonatal sepsis cases yearly worldwide, accounting for more than 3.40% [CrI: 0.75 to 8.01] of all neonatal deaths. The largest relative benefits are in Africa (Sierra Leone, Mali, Niger) and South-East Asia (Bangladesh) where vaccination could avert over 6% of all neonatal deaths. Nevertheless, our modeling only considers country-level trends in K. pneumoniae neonatal sepsis deaths and is unable to consider within-country variability in bacterial prevalence that may impact the projected burden of sepsis. CONCLUSIONS: A K. pneumoniae maternal vaccine could have widespread, sustained global benefits as AMR in K. pneumoniae continues to increase.

The evolution of colistin resistance increases bacterial resistance to host antimicrobial peptides and virulence.

Antimicrobial peptides (AMPs) offer a promising solution to the antibiotic resistance crisis. However, an unresolved serious concern is that the evolution of resistance to therapeutic AMPs may generate cross-resistance to host AMPs, compromising a cornerstone of the innate immune response. We systematically tested this hypothesis using globally disseminated mobile colistin resistance (MCR) that has been selected by the use of colistin in agriculture and medicine. Here, we show that MCR provides a selective advantage to Escherichia coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance. Moreover, MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model. Our study shows how the anthropogenic use of AMPs can drive the accidental evolution of resistance to the innate immune system of humans and animals. These findings have major implications for the design and use of therapeutic AMPs and suggest that MCR may be difficult to eradicate, even if colistin use is withdrawn.

International manufacturing and trade in colistin, its implications in colistin resistance and One Health global policies: a microbiological, economic, and anthropological study.

BACKGROUND: The emergence of colistin-resistant Enterobacterales is a global public health concern, yet colistin is still widely used in animals that are used for food as treatment, metaphylaxis, prophylaxis, and growth promotion. Herein, we investigate the effect of colistin-resistant Enterobacterales in Pakistan, global trade of colistin, colistin use at the farm level, and relevant socioeconomic factors. METHODS: We conducted a microbiological, economic, and anthropological study of colistin-resistant Escherichia coli in humans, animals, and the environment and international trade and knowledge of colistin in Pakistan, Bangladesh, Nigeria, China, India, and Viet Nam. We collected backyard poultry cloacal swabs, commercial broiler cloacal swabs, cattle and buffalo rectal swabs, human rectal swabs, wild bird droppings, cattle and buffalo meat, sewage water, poultry flies, chicken meat, and canal water from 131 sites across Faisalabad, Pakistan, to be tested for mcr-1-positive and mcr-3-positive Escherichia coli. We recruited new patients admitted to Allied Hospital, Faisalabad, Pakistan, with abdominal pain and diarrhoea for rectal swabs. Patients with dysentery and those who were already on antibiotic treatment were excluded. Data for colistin trade between 2017 and 2020, including importation, manufacturing, and usage, were accessed from online databases and government sources in Pakistan, Bangladesh, and Nigeria. We recruited participants from poultry farms and veterinary drug stores in Pakistan and Nigeria to be interviewed using a structured questionnaire. International manufacturing, import, and export data; value analysis; and trade routes of colistin pharmaceutical raw material (PRM), feed additive, and finished pharmaceutical products (FPPs) were accessed from 2017-21 export data sets. FINDINGS: We collected 1131 samples between May 12, 2018, and July 1, 2019: backyard poultry cloacal swabs (n=100), commercial broiler cloacal swabs (n=102), cattle and buffalo rectal swabs (n=188), human rectal swabs (n=200), wild bird droppings (n=100), cattle and buffalo meat (n=100), sewage water (n=90), poultry flies (n=100), chicken meat (n=100), and canal water (n=51). We recruited 200 inpatients at Allied Hospital, Faisalabad, Pakistan, between Nov 15, 2018, and Dec 14, 2018, for rectal swabs. We recruited 21 participants between Jan 1, 2020, and Dec 31, 2020, from poultry farms and drug stores in Pakistan and Nigeria to be interviewed. 75 (7%) of 1131 samples contained mcr-1-positive E coli, including wild bird droppings (25 [25%] of 100), commercial broiler cloacal swabs (17 [17%] of 100), backyard poultry cloacal swabs (one [1%] of 100), chicken meat (13 [13%] of 100), cattle and buffalo meat (two [2%] of 100), poultry flies (eight [8%] of 100), sewage water (six [7%] of 90), and human rectal swabs (three [2%] of 200). During 2017-20, Pakistan imported 275·5 tonnes (68·9 tonnes per year, 95% CI 41·2-96·6) of colistin as PRM, all sourced from China, 701·9 tonnes (175·5 tonnes per year, 140·9-210·1) of colistin as feed additives from China and Viet Nam, and 63·0 tonnes (15·8 tonnes per year, 10·4-21·1) of colistin as FPPs from various countries in Asia and Europe. For Bangladesh and Nigeria, colistin PRM and FPPs were imported from China and Europe. Colistin knowledge and usage practices in Pakistan and Nigeria were unsatisfactory in terms of understanding of the effects on human medicine and usage other than for treatment purposes. China is the major manufacturer of PRM and feed additive colistin and exported a total of 2664·8 tonnes (666·2 tonnes per year, 95% CI 262·1 to 1070·2) of PRM and 2570·2 tonnes (642·6 tonnes per year, -89·4 to 1374·5) of feed additive in 1330 shipments during 2018-21 to 21 countries. INTERPRETATION: Regardless of 193 countries signing the UN agreement to tackle antimicrobial resistance, trading of colistin as PRM, FPPs, and feed additive or growth promoter in low-income and middle-income countries continues unabated. Robust national and international laws are urgently required to mitigate the international trade of this antimicrobial listed on WHO Critically Important Antimicrobials for Human Medicine. FUNDING: Pakistan Agricultural Research Council and INEOS Oxford Institute for Antimicrobial Research TRANSLATION: For the Urdu translation of the abstract see Supplementary Materials section.

Genomic Insights Into the Mechanism of Carbapenem Resistance Dissemination in Enterobacterales From a Tertiary Public Heath Setting in South Asia.

SUMMARY: 10.6% patients were CRE positive. Only 27% patients were prescribed at least 1 antibiotic to which infecting pathogen was susceptible. Burn and ICU admission and antibiotics exposures facilitate CRE acquisition. Escherichia coli ST167 was the dominant CRE clone. BACKGROUND: Given the high prevalence of multidrug resistance (MDR) across South Asian (SA) hospitals, we documented the epidemiology of carbapenem-resistant Enterobacterales (CRE) infections at Dhaka Medical College Hospital between October 2016 and September 2017. METHODS: We enrolled patients and collected epidemiology and outcome data. All Enterobacterales were characterized phenotypically and by whole-genome sequencing. Risk assessment for the patients with CRE was performed compared with patients with carbapenem-susceptible Enterobacterales (CSE). RESULTS: 10.6% of all 1831 patients with a clinical specimen collected had CRE. In-hospital 30-day mortality was significantly higher with CRE [50/180 (27.8%)] than CSE [42/312 (13.5%)] (P = .001); however, for bloodstream infections, this was nonsignificant. Of 643 Enterobacterales isolated, 210 were CRE; blaNDM was present in 180 isolates, blaOXA-232 in 26, blaOXA-181 in 24, and blaKPC-2 in 5. Despite this, ceftriaxone was the most commonly prescribed empirical antibiotic and only 27% of patients were prescribed at least 1 antibiotic to which their infecting pathogen was susceptible. Significant risk factors for CRE isolation included burns unit and intensive care unit admission, and prior exposure to levofloxacin, amikacin, clindamycin, and meropenem. Escherichia coli ST167 was the dominant CRE clone. Clustering suggested clonal transmission of Klebsiella pneumoniae ST15 and the MDR hypervirulent clone, ST23. The major trajectories involved in horizontal gene transfer were IncFII and IncX3, IS26, and Tn3. CONCLUSIONS: This is the largest study from an SA public hospital combining outcome, microbiology, and genomics. The findings indicate the urgent implementation of targeted diagnostics, appropriate antibiotic use, and infection-control interventions in SA public institutions.

Genomic Shift in Population Dynamics of mcr-1-positive Escherichia coli in Human Carriage.

Emergence of the colistin resistance gene, mcr-1, has attracted worldwide attention. Despite the prevalence of mcr-1-positive Escherichia coli (MCRPEC) strains in human carriage showing a significant decrease between 2016 and 2019, genetic differences in MCRPEC strains remain largely unknown. We therefore conducted a comparative genomic study on MCRPEC strains from fecal samples of healthy human subjects in 2016 and 2019. We identified three major differences in MCRPEC strains between these two time points. First, the insertion sequence ISApl1 was often deleted and the percentage of mcr-1-carrying IncI2 plasmids was increased in MCRPEC strains in 2019. Second, the antibiotic resistance genes (ARGs), aac(3)-IVa and blaCTX-M-1, emerged and coexisted with mcr-1 in 2019. Third, MCRPEC strains in 2019 contained more virulence genes, resulting in an increased proportion of extraintestinal pathogenic E. coli (ExPEC) strains (36.1%) in MCRPEC strains in 2019 compared to that in 2016 (10.5%), implying that these strains could occupy intestinal ecological niches by competing with other commensal bacteria. Our results suggest that despite the significant reduction in the prevalence of MCRPEC strains in humans from 2016 to 2019, MCRPEC exhibits increased resistance to other clinically important ARGs and contains more virulence genes, which may pose a potential public health threat.

Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries.

BACKGROUND: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). METHODS: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015-2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. RESULTS: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. CONCLUSIONS: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome.

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study.

BACKGROUND: Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. METHODS: The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. FINDINGS: Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69-234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04-74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37-2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. INTERPRETATION: Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. FUNDING: Bill & Melinda Gates Foundation.

Comparing Long-Read Assemblers to Explore the Potential of a Sustainable Low-Cost, Low-Infrastructure Approach to Sequence Antimicrobial Resistant Bacteria With Oxford Nanopore Sequencing.

Long-read sequencing (LRS) can resolve repetitive regions, a limitation of short read (SR) data. Reduced cost and instrument size has led to a steady increase in LRS across diagnostics and research. Here, we re-basecalled FAST5 data sequenced between 2018 and 2021 and analyzed the data in relation to gDNA across a large dataset (n = 200) spanning a wide GC content (25-67%). We examined whether re-basecalled data would improve the hybrid assembly, and, for a smaller cohort, compared long read (LR) assemblies in the context of antimicrobial resistance (AMR) genes and mobile genetic elements. We included a cost analysis when comparing SR and LR instruments. We compared the R9 and R10 chemistries and reported not only a larger yield but increased read quality with R9 flow cells. There were often discrepancies with ARG presence/absence and/or variant detection in LR assemblies. Flye-based assemblies were generally efficient at detecting the presence of ARG on both the chromosome and plasmids. Raven performed more quickly but inconsistently recovered small plasmids, notably a ∼15-kb Col-like plasmid harboring bla KPC . Canu assemblies were the most fragmented, with genome sizes larger than expected. LR assemblies failed to consistently determine multiple copies of the same ARG as identified by the Unicycler reference. Even with improvements to ONT chemistry and basecalling, long-read assemblies can lead to misinterpretation of data. If LR data are currently being relied upon, it is necessary to perform multiple assemblies, although this is resource (computing) intensive and not yet readily available/useable.

Silent circulation of BKC-1-producing Klebsiella pneumoniae ST442: molecular and clinical characterization of an early and unreported outbreak.

OBJECTIVE: To describe the undetected circulation of an epidemic BKC-1-producing Klebsiella pneumoniae ST442 clone, occasioning the first reported outbreak of the infrequent carbapenemase BKC-1. METHODS: Six hundred and forty-seven K. pneumoniae isolates (2008-2017) with reduced susceptibility to carbapenems were screened for blaBKC-1. BKC-1-positive isolates were typed using pulsed-field gel electrophoresis and multi-locus sequence typing. Susceptibility profiles were determined by broth microdilution, and additional antimicrobial resistance genes (ARGs) were investigated by polymerase chain reaction. Some isolates were submitted to full genomic characterization by whole-genome sequencing (Illumina MiSeq and MinIon), and in-vivo virulence studies using the Galleria mellonella model. RESULTS: Sixteen (2.5%) K. pneumoniae, from 15 patients, carrying blaBKC-1 were found between 2010 and 2012. Among these patients, the all-cause mortality rate was 54.5%. A major clone - A1-ST442 (13/16) - was isolated during the study period. The BKC-1-producing isolates had a multi-drug-resistant phenotype, remaining susceptible to gentamicin (87.5%) and ceftazidime-avibactam (100%) alone. The presence of two carbapenemases - blaBKC-1 and blaKPC-2 - was detected in six isolates, increasing the ß-lactam minimum inhibitory concentration significantly. Additionally, other ARGs were identified on A1-ST442 and B1-ST11 clones. The B1-ST11 clone was more virulent than the A1-ST442 clone. CONCLUSION: An undetected outbreak caused predominantly by a BKC-1-positive A1-ST442 clone between 2010 and 2012 was identified 10 years later in a Brazilian hospital. The misidentification of BKC-1 may have worsened the spread of resistant clones; this reinforces the need for correct and rapid identification of antimicrobial resistance mechanisms in hospitals.

Early-Onset Neonatal Sepsis in Low- and Middle-Income Countries: Current Challenges and Future Opportunities.

Neonatal sepsis is defined as a systemic infection within the first 28 days of life, with early-onset sepsis (EOS) occurring within the first 72h, although the definition of EOS varies in literature. Whilst the global incidence has dramatically reduced over the last decade, neonatal sepsis remains an important cause of neonatal mortality, highest in low- and middle-income countries (LMICs). Symptoms at the onset of neonatal sepsis can be subtle, and therefore EOS is often difficult to diagnose from clinical presentation and laboratory testing and blood cultures are not always conclusive or accessible, especially in resource limited countries. Although the World Health Organisation (WHO) currently advocates a ß-lactam, and gentamicin for first line treatment, availability and cost influence the empirical antibiotic therapy administered. Antibiotic treatment of neonatal sepsis in LMICs is highly variable, partially caused by factors such as cost of antibiotics (and who pays for them) and access to certain antibiotics. Antimicrobial resistance (AMR) has increased considerably over the past decade and this review discusses current microbiology data available in the context of the diagnosis, and treatment for EOS. Importantly, this review highlights a large variability in data availability, methodology, availability of diagnostics, and aetiology of sepsis pathogens.